Wound healing is a complex biological process that involves many different cell types, many different cytokines, the extracellular matrix (ECM), and numerous interactions among them. Most wounds heal rapidly and efficiently within a week or two; however, the result is neither aesthetically nor functionally perfect. Wound contraction and scar formation are currently unavoidable results of wound healing. Scar tissue is less flexible than normal skin and can be cosmetically disfiguring, and wound contraction can lead to joint disablement (Lamme et al., J. Histochem. Cytochem. 44:1311, 1996). Scars lack elastin and consist of poorly reconstituted collagen matrix in dense parallel bundles rather than the mechanically efficient basket-weave meshwork of collagen in unwounded dermis (Martin, Science 276:75, 1997). Two major goals of wound-healing biology are more rapid wound healing and more perfect reconstruction of the damaged parts. Compositions and methods useful in accomplishing these goals are currently needed.
Wound Healing
Wound healing has been divided into a number of overlapping phases. These include fibrin clot formation, recruitment of inflammatory cells, reepitheliazation, and matrix formation and remodeling. Immediately after tissue injury, blood vessel disruption leads to the extravasation of blood and concomitant platelet aggregation and blood coagulation resulting in fibrin clot formation. Activated platelets trapped within the fibrin clot degranulate and release a variety of cytokines and growth hormones. These cytokines and growth hormones help to recruit inflammatory cells to the site of injury, to stimulate angiogenesis, and to initiate the tissue movements associated with reepitheliazation and connective tissue contraction.
Neutrophils and monocytes are recruited to the site of injury by a number of chemotactic signals including the growth factors and cytokines released by the degranulating platelets, formyl methionyl peptides cleaved from bacterial proteins, and the by-products of proteolysis of fibrin and other matrix proteins. Neutrophil infiltration ceases after a few days, but macrophages continue to accumulate by continued recruitment of monocytes to the wound site. Activated macrophages release growth factors and cytokines thereby amplifying the earlier signals from the degranulating platelets.
Formation of granulation tissue and reepithelialization of the wound site begins after several hours (Clark, J. Am. Acad. Dermatol. 13:701, 1985). Reepithelialization is performed by the basal keratinocytes which lose their attachments to the basal lamina and crawl over the provisional matrix of fibrin and fibronectin, and underlying matrix. Some hours after the onset of migration, epidermal cells begin to reproduce and thereby provide the cells needed to replace those lost during the injury. Keratinocyte proliferation is regulated by keratinocyte growth factor and members of the epidermal growth factor (EGF) family. In order to migrate through the fibrin clot, the keratinocytes must dissolve the fibrin barrier in front of them. Plasmin is the chief fibrinolytic enzyme used in this process. Reepitheliazation is made easier by the underlying contractile connective tissue, which shrinks to bring the wound margins toward one another. Epidermal migration ceases when the wound surface has been covered by a monolayer of cells.
Cells of the new epidermis undergo the standard differentiation program of cells in the outer layers of unwounded epidermis. A new stratified epidermis is, thereby, reestablished from the margins of the wound inward. Matrix formation and remodeling begins simultaneously with reepithelialization. The matrix is constantly altered over the next several months with the elimination of the fibronectin from the matrix and the accumulation of collagen that provides the residual scar with increasing tensile strength. Elastin fibers, which are responsible for the elasticity of tissue, are only detected in human scars years after the injury (Compton et al., Lab. Invest. 60:600, 1989).
Methods to Promote Wound Healing
In the past, many methods have been proposed and tested to promote wound healing and limit scarring; however, better methods and compositions are still needed. These older methods include cyanoacrylate tissue adhesives, a combination of epidermal transplantation and a collagen/elastin dermal substitute, application of collagen and glycosaminoglycans to the site of injury, and biocompatible adhesives with collagen.
One of the more popular methods is the use of cyanoacrylate tissue adhesives. These adhesive have been used in place of and in conjunction with sutures. Cyanoacrylate adhesives have been used in cases ranging from cardiac surgery (Robicsek et al., J. Card. Surg. 9:353, 1994) to simple lacerations in the pediatric population (Penoff, Plast. Reconstr. Surg. 103:730, 1999). One study has found that cyanoacrylate tissue adhesive may be the preferred method in terms of cosmetic appearance for the cutaneous closure of facial lacerations oriented against Langer""s lines (Simon et al., J. Emerg. Med. 16:185, 1998).
In a study of the healing of full-thickness wounds in pigs, a dermal matrix consisting of native bovine collagen coated with elastin hydrolysate was found to serve as a template for dermal tissue regeneration in combination with an epidermal transplantation. This combination treatment reduced wound contraction and improved tissue regeneration (Lamme et al., J. Histochem. Cytochem. 44:1311, 1996).
U.S. Pat. No. 4,837,024, issued Jun. 6, 1989, to Michaeli et al., discloses an article to promote healing of a surface wound. A suspension of particles of collagen and a glycosaminoglycan is contacted with the wound surface. Collagen is a major component of the ECM and helps to promote wound healing. The glycosaminoglycan is chemotactic of fibroblasts and/or endothelial cells. The collagen/glycosaminoglycan is applied to the wound and maintained in contact with the wound for an extended period of time, i.e., during the entire healing process or until at least closure of the wound by new tissue. The application promotes the vascularization of the wound, attracts fibroblasts and endothelial cells, and generally provides a favorable environment for the cells during the healing process.
U.S. Pat. No. 5,614,587, issued Mar. 25, 1997, to Rhee et al., and U.S. Pat. No. 5,744,545, issued Apr. 28, 1998, to Rhee et al., disclose a composition suitable for use as a bioadhesive and a method for using such a composition. The composition comprises fibrillar collagen, a fiber disassembly agent, and a multifunctionally activated synthetic hydrophilic polymer. The collagen and polymer are mixed to initiate cross-linking, the collagen-polymer mixture is then applied to a first surface before substantial cross-linking has occurred, and then a second surface is brought in contact with the first surface. The composition is optically clear so that it could be used in ophthalmic applications, and the composition comprises biocompatible, non-immunogenic components which leave no toxic, potentially inflammatory or immunogenic reaction products at the tissue site of administration.
An invention which would promote healing and lessen scarring would be of great value.
The present invention provides compositions and methods useful in the promotion of wound healing. These compositions comprise virgin monomers of tropoelastin and the cross-linking enzyme, lysyl oxidase. The method comprises mixing these two components of the composition together and applying them to a wound before substantial cross-linking has occurred. The tropoelastin monomers and lysyl oxidase only come in contact with each other immediately before application to the wound or during application to the wound.
Without wishing to be bound by any particular theory, we propose that the lysyl oxidase catalyzes the oxidative deamination of the lysine residues of the tropoelastin monomers at the site of the wound. Then in a non-enzymatic step, cross-links form between the tropoelastin monomers as well as between tropoelastin monomers and other proteins of the extracellular matrix such as collagen. The cross-linked elastin at the site of injury helps to hold the injured tissue together and thereby promotes healing. The elastin is also chemotactic for fibroblasts, endothelial cells, and inflammatory cells, thereby promoting healing in another manner. Elastin at the site of injury also helps to lessen scarring since scar tissue is devoid of elastin, and elastin is an important component of uninjured skin. The cross-linked elastin also generally provides a favorable environment for the cells that participate in the healing process.
In preferred embodiments of the invention, one or both of the tropoelastin monomers and lysyl oxidase are made recombinantly and purified to homogeneity using standard techniques. The purified tropoelastin and lysyl oxidase may then be suspended in a liquid, such as an aqueous solution (e.g., water or saline) or an organic solvent, or provided in a dry powder form, or in a lyophilized form. These two components, tropoelastin and lysyl oxidase, are kept separate from each other until right before use. In another embodiment, the lysyl oxidase is kept in an inactive from in the presence of the tropoelastin, and the lysyl oxidase not activated until right before use. In yet another embodiment, the two components are applied to the wound separately.
In the method of the present invention, the composition may be applied only once at the time of the injury or more than once over the course of wound healing. The composition of tropoelastin and lysyl oxidase may also be used in conjunction with sutures, staples, or adhesive strips in closing the wound.
The composition of the present invention may also be used in promoting the healing of wounds involving structures with elastic fibers such as arteries, lung tissue, or skin. In particular, the composition may be used in surgeries involving arteries, lungs, or the skin.
In another embodiment of the present invention, the tropoelastin comprises only portions of the tropoelastin protein, preferably containing at least one cross-linking domain. In another embodiment, the lysyl oxidase comprises only an active portion of the enzyme.
The preferred compositions of the present invention are biocompatible, non-toxic, and non-immunogenic, and potentially inflammatory or immunogenic reaction products at the site of administration are avoided.